A technique for the measurement of GFR without collection of urine in rats was experimentally validated and applied to experiments designed to: (1) evaluate the degree of reduction of GFR in rats with congenital, unilateral hydronephrosis; and (2) to determine if the reduction in renal function is mediated by angiotensin II and/or thromboxane A2 mechanisms. Simultaneous measurements of GFR by a constant-infusion technique and the traditional inulin clearance technique in rats with either one or two normal kidneys were highly correlated (r = 0.934; P < 0.001; N = 17). GFR was approximately 24% lower (P < 0.001) in rats with congenital unilateral hydronephrosis than in rats with a normal kidney. The GFR in rats with hydronephrosis infused with a receptor blocker for either angiotensin II or thromboxane A2 was greater than the GFR in hydronephrotic kidneys without blockade and was not significantly different (P > 0.05) from that in rats with normal kidneys. These results indicate that a constant inulin infusion technique without urine collections can be used to accurately measure GFR in congenitally hydronephrotic kidneys, rendering values free from possible residual pelvic volume artifact. In addition, these results also indicate that a significant 24% reduction in GFR occurs in congenital unilateral hydronephrosis and is mediated by angiotensin II and thromboxane A2 mechanisms.